Improving spinosad production by tuning expressions of the forosamine methyltransferase and the forosaminyl transferase to reduce undesired less active byproducts in the heterologous host Streptomyces albus J1074

Background Spinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-o-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad. Results By tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%. Conclusion N-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02023-3.


Background
Spinosad, the mixture of spinosyns A and D, is a fermentation derived insecticide with high activity against a variety of chewing insect pests and exceptional safety profiles [1,2]. During spinosad biosynthesis, a linear polyketide chain is made first by polyketide synthases SpnA-E [3], and then the tetracyclic aglycone is formed by cross-bridging enzymes SpnF, J, L, M [4]. Rhamnose is biosynthesized from glucose-1-phosphate by Gtt, Gdh, Epi, and Kre proteins, added to the aglycone by the transferase SpnG [5] and then o-methylated by SpnH, I, K [6] to form the pseudoaglycone. Forosamine biosynthesis shares the common first two steps with rhamnose [7] producing TDP-4-keto-6-deoxy-d-glucose which is further converted into TDP-d-forosamine by SpnO, N, Q, R, and S. N-methylation of the forosamine catalyzed by SpnS occurs in a stepwise manner and the monomethylated product (N-monodesmethyl forosamine) can be released from the active site of SpnS [8]. Forosamine is transferred onto the pseudoaglycone by SpnP [9] to finally form spinosad. SpnP can also transfer N-monodesmethyl and N,N-didesmethyl forosamines to the pseudoaglycone to produce N-monodesmethyl and N,N-didesmethyl spinosad [8] (Fig. 1). The insecticidal activities of N-monodesmethyl and N,N-didesmethyl spinosad are both lower than spinosad. The LC 50 of spinosyn A to Heliothis virescens larvae are 0.3 mg L − 1 , while the LC 50 of N-monodesmethyl spinosyn A (spinosyn B) and N,N-didesmethyl spinosyn A (spinosyn C) are 0.4 mg L − 1 and 0.8 mg L − 1 respectively [10]. The LC 50 of N-monodesmethyl spinosyn D to Heliothis virescens larvae is seven times higher than that of spinosyn D (5.6 vs. 0.8 mg L − 1 ) [10]. Therefore, N-desmethyl spinosad is undesired byproduct in the fermentation.
Because the spinosad native producer Saccharopolyspora spinosa is difficult to genetically manipulate, its gene cluster has been cloned and expressed in heterologous hosts such as S. erythraea [11], S. coelicolor [12], and S. albus [13,14]. Previously, we constructed an artificial gene cluster in which the 23 spinosad biosynthetic genes were grouped into 7 operons under control of Streptomyces constitutive strong promoters [14]. When the artificial gene cluster was expressed in S. albus J1074, the yield of spinosad was improved 328 folds compared with the native gene cluster. Besides spinosad, the less active byproduct N-monodesmethyl spinosad was also detected at the same titer as spinosad in the fermentation broth of S. albus J1074 containing the artificial gene cluster.
Tuning expression of biosynthetic genes is an important way to reduce undesired compound production or enhance desired compound production. For example, in erythromycin A fermentation, erythromycins B and C are recognized as undesired byproducts because they are much less active and cause greater side effects [15]. Chen et al. nearly completely eliminated erythromycins B and C and improved the titer of erythromycin A by 25% by tuning expression of two tailoring enzymes, the P450 hydroxylase EryK and S-adenosylmethioninedependent O-methyltransferase EryG [16]. In this study, we revealed that N-desmethyl spinosad was produced due to unbalanced expression of the forosamine methyltransferase gene spnS and upstream biosynthetic genes which caused accumulation of N-desmethyl forosamine. By tuning expression of spnS, we reduced the content of undesired less active byproduct N-desmethyl spinosad by more than 90% in the fermentation broth of S. albus J1074. Furthermore, the yield of desired product spinosad was increased by 5.3 folds to 5.8 mg L − 1 through overexpressing spnS together with the forosamyltransferase gene spnP.

Results and discussion
Expression of the forosamine methyltransferase gene spnS affected production of spinosad and its N-desmethyl derivatives Previously, we constructed an artificial gene cluster for efficient heterologous spinosad production in S. albus J1074 [14]. The artificial gene cluster consisted of all 23 spinosad biosynthetic genes grouped into 7 operons under control of strong constitutive promoters. In the fermentation broth of S. albus J1074 harboring the 7-operon artificial gene cluster (7op), besides the desired product spinosad, the less active analogue N-monodesmethyl spinosad was also detected (Fig. 2). The titer of N-monodesmethyl spinosyn A (spinosyn B) is the same as that of spinosyn A. In the forosamine biosynthesis, dimethylation of its amino group by SpnS occurs in a stepwise manner. The intermediates N,N-didesmethyl forosamine and N-monodesmethyl forosamine can also be transferred onto the macrolide backbone by SpnP. We speculated that the N-monodesmethyl spinosad was produced due to accumulation of N-monodesmethyl forosamine caused by insufficient expression of SpnS.
To determine the effects of spnS expression level on biosynthesis of spinosad and its N-desmethyl derivatives, we inserted the cumate-inducible cymR-P21-cmt expression system [18] upstream of the spnS gene in the artificial gene cluster by recombineering to generate the cum-spnS recombinant gene cluster (Fig. 3A). Recombineering is a DNA engineering technique which uses homologous recombination mediated by phage proteins in E. coli [19]. In the cymR-P21-cmt expression system, the CmyR repressor binding on the cmt operator placed downstream of the P21 promoter will block transcription of downstream genes. The inducer cumate added in the medium can bind CmyR and restore the transcription.
During fermentation of S. albus J1074 containing the cum-spnS gene cluster, when no cumate inducer was added into the medium, spinosyn A, N-monodesmethyl spinosyn A (spinosyn B), and N,N-didesmethyl spinosyn A (spinosyn C) were all produced. The titer of spinosyn B is higher than that of spinosyn A which is higher than that of spinosyn C ( Fig. 3B and Additional file 1: Fig. S1). Because the cymR-P21-cmt promoter is not completely tight [20], low level leaky expression of SpnS caused production of N-monodesmethyl forosamine and N,Ndidesmethyl forosamine which were both transferred onto the macrolide backbone by SpnP. As the cumate  [17] concentration in the fermentation broth increased, titers of spinosyns B and C decreased. When more than 20 µM of cumate was added into the fermentation medium, production of spinosyn C cannot be detected and production of spinosyn B decreased by more than 87% compared with the production without cumate addition. The lowest spinosyn B production, decreased by 98%, was obtained when 50 µM of cumate was added ( Fig. 3B and Additional file 1: Fig. S1 and Table S1).
As the production of spinosyns B and C decreased, the production of spinosyn A increased when less than 20 µM of cumate was added. However, when more cumate was added, the yield of spinosyn A did not increase further as the production of spinosyns B and C decreased.

Constitutive overexpression of spnS reduced production of N-desmethyl spinosyn A derivatives and improved spinosyn A production
In the cumate-inducible cymR-P21-cmt expression system, production of spinosyn A and its N-desmethyl derivatives was depended on the spnS expression level. We then replace the inducible cymR-P21-cmt expression system upstream of spnS with the strong constitutive kasOp* promoter [21] to generate the kas-spnS recombinant gene cluster (Fig. 4A). When the kas-spnS gene cluster was introduced into S. albus J1074, production of spinosyn A and its desmethyl derivatives are similar with the inducible cymR-P21-cmt expression system at high cumate concentration. Production of spinosyn C cannot be detected and production of spinosyn B decreased by 86% compared with the production from the original 7op gene cluster. The spinosyn A production from the kas-spnS gene cluster increased only 38% compared with the original 7op gene cluster (Fig. 4B).

Constitutive overexpression of the forosamyltransferase gene spnP together with spnS further improved spinosyn A production
Above results suggested that although the amino group of forosamine was almost fully methylated in the spnS overexpression strain, a large quantity of fully methylated forosamine was not used for the biosynthesis of spinosad. We speculated that expression of the forosamyltransferase SpnP was not sufficient for fully transfer of forosamine to the spinosad pseudoaglycone. Two strong constitutive promoters (kasOp*, SA15p [22]) were inserted upstream of spnP in the kas-spnS gene cluster to . Error bars represent standard deviation. Differences were analyzed by one-way ANOVA and P < 0.05 was considered statistically significant. ***P < 0.001, **P < 0.01, *P < 0.05 generate recombinant gene clusters kas-spnP-kas-spnS and SA15-spnP-kas-spnS (Fig. 5A).
When the kas-spnP-kas-spnS and SA15-spnP-kas-spnS gene clusters were introduced into S. albus J1074 respectively, production of spinosyn C (N,N-didesmethyl spinosyn A) was not detected and production of spinosyn B (N-monodesmethyl spinosyn A) was low and similar with that in the kas-spnS strain (Fig. 5B). The titers of spinosyn A in the kas-spnP-kas-spnS and SA15-spnP-kas-spnS strains were 4.4 and 3.1 times higher than that in the kas-spnS strain, respectively. Finally, the spinosad (spinosyns A and D) production in S. albus J1074 was increased to 5.8 ± 0.4 mg L − 1 when both spnS and spnP were overexpressed under control of the kasOp* promoter (Fig. 5 C). This suggested that enhanced expression of SpnP significantly promoted transfer of forosamine to the pseudoaglycone and make the spinosad biosynthesis much more efficient.

Conclusion
A polyketide assembly line often produces multiple structurally related compounds, such as avermectins [23], erythromycins [15] and spinosyns [2]. Biosynthesis of less active components will compete substrates and energy with the most active components, therefore, eliminating the production of byproducts is important for enhancing the titer of the desired compounds. In this study, we improved spinosad production by tuning Fig. 4 Effects of constitutive spnS overexpression on productions of spinosyns A and B. A Schematic representation of the kasOp*-spnS expression system. B Titers of spinosyns A and B from different gene clusters. Each fermentation was done in triplicate (n = 3). Error bars represent standard deviation. Differences were analyzed by one-way ANOVA and P < 0.05 was considered statistically significant. ***P < 0.001, **P < 0.01, *P < 0.05

Fig. 5
Effects of both spnS and spnP overexpression on spinosyn A and its N-desmethyl derivatives. A Schematic representation of the kas-spnP-kas-spnS and SA15-spnP-kas-spnS expression system. B Yield of spinosyn B from different gene clusters. C Yield of spinosad (spinosyns A and D) from different gene clusters. Each fermentation was done in triplicate (n = 3). Error bars represent standard deviation. Differences were analyzed by one-way ANOVA and P < 0.05 was considered statistically significant. ***P < 0.001, **P < 0.01, *P < 0.05 expressions of the forosamine methyltransferase and the forosaminyl transferase to reduce undesired less active N-desmethyl byproducts in the heterologous host S. albus J1074. On the other hand, fine tuning expression of tailoring enzymes can channel the biosynthesis to specific analogues such as them with different methylation status which is helpful for diversification of structures.
Gene cluster reconstruction has been widely employed in optimizing production of natural products [24][25][26]. The less active N-desmethyl spinosad byproduct was accumulated due to unbalanced expression of the forosamine methyltransferase gene and upstream biosynthetic genes when we refactored the spinosad gene cluster. Therefore, balanced expression of biosynthetic genes should be considered in the reconstruction strategy to avoid accumulation of undesired intermediates or analogues.
Above constructed spinosad expression vectors were transformed into S. albus J1074 by conjugation [28] for fermentation and high performance liquid chromatography-mass spectrometry analysis.
Additional file 1: Figure S1. SHPLC-MS Analysis (Base Peak Chromatogram) of Streptomyces albus J1074 with pBAC-spnNEW-cum-spnS under different cumate concentrations. Figure S2. Restriction analysis of recombinant BACs in this work. Table S1. Production of spinosyns A, B and C in Streptomyces albus J1074 with pBAC-spnNEW-cum-spnS under different cumate concentrations; Table S2. Strains and plasmids used in this work. Table S3. Primers used in this work.